Radiolabeled compounds for thrombus imaging

ABSTRACT

This invention relates to radiolabeled scintigraphic imaging agents, and methods and reagents for producing such agents. Specifically, the invention relates to specific binding compounds, including peptides, that bind to a platelet receptor that is the platelet GPIIb/IIIa receptor, methods and kits for making such compounds, and methods for using such compounds labeled with technetium-99m via a covalently-linked radiolabel-binding moiety to image thrombi in a mammalian body.

This application is a 371 of PCT/US94/03878, filed April 8, 1994, whichclaims priority to U.S. patent application Ser. No. 08/044,825, filedApr. 8, 1993 and now abandoned; this application is also acontinuation-in-part of U.S. patent application Ser. No. 08/439,905,filed May 12, 1995 and now U.S. Pat. No. 5,645,815; which is acontinuation of U.S. patent application Ser. No. 08/044,825, filed Apr.8, 1993 and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to scintigraphic imaging agents and reagents, andmethods for producing such agents and reagents. Specifically, theinvention relates to reagents that can be radiolabeled withtechnetium-99m (Tc-99m), methods and kits for making and radiolabelingsuch reagents, and methods for using such radiolabeled reagents to imagesites of thrombus formation in a mammalian body.

2. Description of the Prior Art

Thrombosis and thromboembolism, in particular deep vein thrombosis (DVT)and pulmonary embolism (PE), are common clinical conditions that areassociated with significant morbidity and mortality. It has beenestimated that in the U.S. approximately 5 million patients experienceone or more episodes of DVT per year and that over 500,000 cases ofpulmonary embolism occur, resulting in 100,000 deaths (J. Seabold,Society of Nuclear Medicine Annual Meeting 1990). It has also beenestimated that over 90% of all pulmonary emboli arise from DVT in thelower extremities. Anticoagulant therapy can effectively treat theseconditions if applied early enough. However, such treatment isassociated with risks (e.g. internal bleeding) that prevent unnecessaryprophylactic application. More advanced techniques of thrombolyticintervention (such as the administration of recombinant tissueplasminogen activator or streptokinase) can be used in acute cases, butthese techniques carry even greater risk. Moreover, effective clinicalapplication of these techniques requires that the site of the offendingthrombus be identified so as to monitor the effect of treatment.

For these reasons, a rapid means of localizing thrombi in vivo, mostpreferably using non-invasive methods, is highly desirable. Methodscurrently utilized for the identification of sites of deep-veinthrombosis are contrast venography and compression B-mode ultrasound;the choice of which technique is used depends on the expected locationof the thrombus. However, the former technique is invasive and bothtechniques are uncomfortable for the patient. In addition, these methodsare in many cases either unsuitable or yield inaccurate results.

Current methods used to diagnose PE include chest X-ray,electrocardiogram (EKG), areterial oxygen tension, perfusion andventilation lung scans, and pulmonary angiography. Apart from the latter(invasive) procedure, none of these methods is capable of providing anunequivocal diagnosis.

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ⁶⁸ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yband ¹⁸⁶ Re. Of these radionuclides, Tc-99m and ¹¹¹ In are preferredsingle photon-emitting radionuclides and ⁶⁸ Ga is preferred as apositron-emitting radionuclide. Tc-99m is a preferred radionuclidebecause it emits gamma radiation at 140 keV, it has a physical half-lifeof 6 hours, and it is readily available on-site using amolybdenum-99/technetium-99m generator.

A gamma-emitting radiotracer that binds specifically to a component of athrombus in preference to other tissues when administered in vivo canprovide an external scintigraphic image which defines the location ofthe thrombus-bound radiotracer and hence the thrombus. Thrombi areconstructs of blood cells, largely activated platelets, enmeshed incross-linked fibrin. Activated platelets are particularly good targetsfor radioimaging thrombi because they are not normally found incirculating blood (which contains unactivated platelets).

Activated platelets express the GPIIb/IIIa receptor on their cellsurfaces. The normal ligand for this receptor is fibrinogen (Plow etal., 1987, Perspectives in Inflammation, Neoplasia and Vascular CellBiology, pp. 267-275). However, small, synthetic analogues, which may bebut are not necessarily peptides, have been developed that bind to thisreceptor (examples include Klein et al., 1992, U.S. Pat. No. 5,086,069and Egbertson et al., 1992, European Patent Application No. EPA0478328A1). Although many of these synthetic molecules bind with onlylow affinity, others have been made that have very high affinity (seeEgbertson et al., ibid.). This invention provides small, synthetic,radiolabeled (preferably Tc-99m, ¹¹¹ In or 68Ga labeled) compounds thatbind to the GPIIb/IIIa receptor with high affinity, as scintigraphicagents for non-invasive imaging of thrombi in vivo.

Attempts to provide radiotracers for imaging thrombi are known in theprior art. These include autologous platelets, labeled with either ¹¹¹In or ^(99m) Tc (Tc-99m), and 123I- and 125I-labeled fibrinogen (thelatter detected with a gamma scintillation probe as opposed to a gammacamera). Additional radiolabeled compounds used to label thrombi includeplasmin, plasminogen activators, heparin, fibronectin, fibrin FragmentE₁ and anti-fibrin and anti-platelet monoclonal antibodies [see Knight,1990, Sem. Nucl. Med. 20: 52-67 for review].

Compounds having the ability to bind to the platelet GPIIb/IIIa receptorare known in the prior art.

Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describe peptides ofsequence X-Arg-Gly-Asp-R-Y, wherein X and Y are either H or an aminoacid, and R is Thr or Cys, the peptides being capable of binding toplatelets.

Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides ofsequence Arg-Gly-Asp-X, wherein X is Ser, Thr or Cys, the peptides beingcapable of binding to platelets.

Klein et al., 1992, U.S. Pat. No. 5,086,069 disclose guanine derivativesthat bind to the GPIIb/IIIa receptor.

Pierschbacher et al., 1989, PCT/US88/04403 discloseconformationally-restricted RGD containing peptides for inhibiting cellattachment to a substratum.

Nutt et al., 1990, European Patent Application 90202015.5 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202030.4 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202031.2 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202032.0 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90311148.2 disclosecyclic peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90311151.6 disclosecyclic peptides that are fibrinogen receptor antagonists.

Ali et al., 1990, European Patent Application 90311537.6 disclose cyclicpeptides that are fibrinogen receptor antagonists.

Barker et al., 1991, PCT/US90/03788 disclose cyclic peptides forinhibiting platelet aggregation.

Pierschbacher et al., 1991, PCT/US91102356 disclose cyclic peptides thatare fibrinogen receptor antagonists.

Duggan et al, 1992, European Patent Application 92304111.5 disclosefibrinogen receptor antagonists.

Garland et al, 1992 European Patent Applications 92103861.8 and92108214.5 disclose phenylamide derivatives as platelet aggregationinhibitors.

Bondinell et al, 1993, International Patent Application Serial No.PCT/US92/05463 disclose bicyclic fibrinogen antagonists.

Blackburn et al., International Patent Application Serial No.PCT/US92/08788, disclose nonpeptidyl integrin inhibitors havingspecificity for the GPIIb/IIIa receptor.

Egbertson et al., 1992, European Patent Application 0478328A1 disclosetyrosine derivatives that bind with high affinity to the GPIIb/IIIareceptor.

Ojima et al., 1992, 204th Meeting, Amer. Chem. Soc. Abst. 44 disclosesynthetic multimeric RDGF peptides useful in inhibiting plateletaggregation.

Hartman et al., 1992, J. Med. Chem. 35: 4640-4642 describe tyrosinederivatives that have a high affinity for the GPIIb/IIIa receptor.

Radiolabeled peptides for radioimaging thrombi have been reported in theprior art.

Stuttle, 1990, PCT/GB90100933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

The use of chelating agents for radiolabeling peptides, and methods forlabeling peptides with Tc-99m are known in the prior art and aredisclosed in co-pending U.S. patent applications Ser. No. 07/653,012,now abandoned, a continuation of which issued as U.S. Pat. No.5,811,394; Ser. No. 07/807,062, now U.S. Pat. No. 5,443,815; Ser. No.07/871,282, a divisional of which issued as U.S. Pat. No. 5,780,007;Ser. No. 07/886,752, now abandoned, a continuation of which issued asU.S. Pat. No. 5,849,260; Ser. No. 07/893,981, now U.S. Pat. No.5,508,020; Ser. No. 07/955,466, now abandoned; Ser. No. 08/019,864, nowU.S. Pat. No. 5,552,525; and Ser. No. 08/073,577, now U.S. Pat. No.5,561,220, and radiolabeled peptides for use as scintigraphic imagingagents for imaging thrombi are known in the prior art and are disclosedin co-pending U.S. patent applications Ser. No. 07/886,752, nowabandoned, a continuation of which issued as U.S. Pat. No. 5,849,260;Ser. No. 07/893,981, now U.S. Pat. No. 5,508,020; and Ser. No.08/044,825, now abandoned, a continuation of which issued as U.S. Pat.No. 5,645,815, which are hereby incorporated by reference.

There remains a need for small (to enhance blood and background tissueclearance), synthetic (to make routine manufacture practicable and toease regulatory acceptance), high-affinity, specific-binding moleculesradiolabeled with a convenient radiolabel, preferably Tc-99m, for use inimaging thrombi in vivo. Small synthetic compounds that bindspecifically to the GPIIb/IIIa receptor on activated platelets, that areradiolabeled with a conventient radioisotope, preferably Tc-99m, ¹¹¹ Inor ⁶⁸ Ga, fulfill this need in the art, and are provided by thisinvention.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic thrombus imaging agentsthat are radioactively-labeled reagents. Specifically, the inventionprovides reagents for preparing thrombus imaging agents that areradiolabeled with technetium-99m (Tc-99m), ¹¹¹ In or ⁶⁸ Ga, preferablywith Tc-99m. The reagents of the invention are each comprised of aspecific binding compound, including but not limited to peptides, thatbinds specifically and with high affinity to the platelet glycoproteinIIb/IIIa (GPIIb/IIIa) receptor, that is covalently linked to aradiolabel-complexing moiety.

For optimal imaging, the reagent must be capable of binding to theplatelet GPIIb/IIIa receptor with sufficient affinity that it inhibitsthe adenosine diphosphate (ADP)-induced aggregation of human plateletsin a standard platelet aggregation assay (see Example 3 below) whenpresent at a concentration of no more than 0.3 μM. Also, it is ofdistinct commercial advantage to use small compounds, preferably havinga molecular weight of less than about 10,000 daltons. Such smallcompounds can be readily manufactured. Moreover, they are likely not tobe immunogenic and to clear rapidly from the vasculature, thus allowingfor better and more rapid imaging of thrombi. In contrast, largermolecules such as antibodies of fragments thereof, or otherbiologically-derived peptides larger than 10,000 daltons, are costly tomanufacture, and are likely to be immunogenic and clear more slowly fromthe bloodstream, thereby interfering with rapid diagnoses of thrombi invivo.

The invention also provides reagents wherein the specific bindingcompounds are linear or cyclic peptides having an amino acid sequence of4 to 100 amino acids.

One aspect of the invention provides a reagent for preparing a thrombusimaging agent that is capable of being radiolabeled for imaging thrombiwithin a mammalian body, comprising a specific binding compound thatspecifically binds to the platelet GPIIb/IIIa receptor, and that iscovalently linked to a Tc-99m complexing moiety of formula:

    C(pgp).sup.s -(aa)-C(pgp).sup.s                            I.

wherein C(pgp)^(s) is a protected cysteine and (aa) is any primary α- orβ-amino acid not containing a thiol group. In a preferred embodiment,the amino acid is glycine.

In another embodiment, the invention provides a reagent for preparing athrombus imaging agent that is capable of being radiolabeled for imagingthrombi within a mammalian body, comprising a specific binding compoundthat specifically binds to the platelet GPIIb/IIIa receptor, that iscovalently linked to a Tc-99m complexing moiety comprising a singlethiol-containing moiety of formula:

    A-CZ(B)-[C(R.sup.1 R.sup.2)].sub.n -X                      II.

wherein A is H, HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acidor peptide)-OOC or R⁴ ; Z is H or R⁴ B is H, SH or --NHR³, --N(R³),--N(R)-(amino acid or peptide) or R⁴ ; X is SH or --NHR³, N(R³)-(aminoacid or peptide) or R⁴ ; R¹, R², R³ and R⁴ are independently H orstraight or branched chain or cyclic lower alkyl; n is 0, 1 or 2; and:(1) where B is --NHR³ or --N(R³)-(amino acid or peptide), X is SH and nis 1 or 2; (2) where X is --NHR or --N(R³)-(amino acid or peptide), B isSH and n is 1 or 2; (3) where B is H or R⁴, A is HOOC, H₂ NOC, (aminoacid or peptide)-NHOC, (amino acid or peptide)-OOC, X is SH and n is 0or 1; (4) where A is H or R⁴, then where B is SH, X is --NHR³ or--N(R³)-(amino acid or peptide) and where X is SH, B is --NHR³ or--N(R³)-(amino acid or peptide); (5) where X is H or R⁴, A is HOOC, H₂NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and B isSH; (6) where Z is methyl, X is methyl, A is HOOC, H₂ NOC, (amino acidor peptide)-NHOC, (amino acid or peptide)-OOC and B is SH and n is 0;and B; and wherein the thiol moiety is in the reduced form and wherein(amino acid) is any primary α- or β-amino acid not containing a thiolgroup.

In particular embodiments of this aspect of the invention, theradiolabel-complexing moiety has a formula that is:

IIa. -(amino acid)¹ -(amino acid)² -{A-CZ(B)-[C(R¹ R²)]_(n) -X},

IIb. -{A-CZ(B)-[C(R¹ R²)]_(n) -X}-(amino acid)¹ -(amino acid)²,

IIc. -(a primary α,ω- or β,ω-diamino acid)-(amino acid)¹ -{A-CZ(B)-[C(R¹R²)]_(n) -X}, or

IId. -{A-CZ(B)-[C(R¹ R²)]_(n) -X}-(amino acid)¹ -(a primary α,β- orα,γ-diamino acid)

wherein (amino acid)¹ and (amino acid)² are each independently anynaturally-ocurring, modified, substituted or altered α- or β-amino acidnot containing a thiol group; A is H, HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC or R⁴ ; Z is H or R⁴ B is H,SH or --NHR³, --N(R³)-(amino acid or peptide) or R⁴ ; X is SH or --NHR³,--N(R³)-(amino acid or peptide) or R⁴ ; R¹, R², R³ and R⁴ areindependently H or straight or branched chain or cyclic lower alkyl; nis an integer that is either 0, 1 or 2; and: (1) where B is --NHR³ or--N(R³)-(amino acid or peptide), X is SH and n is 1 or 2; (2) where X is--NHR³ or --N(R³)-(amino acid or peptide), B is SH and n is 1 or 2; (3)where B is H or R⁴, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC, X is SH and n is 0 or 1; (4) where A is Hor R⁴, then where B is SH, X is --NHR³ or --N(R³)-(amino acid orpeptide) and where X is SH, B is --NHR³ or --N(R³)-(amino acid orpeptide); (5) where X is H or R⁴, A is HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC and B is SH; (6) where Z ismethyl, X is methyl, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC and B is SH and n is 0; and (7) where B isSH and X is SH, n is not 0; and wherein the thiol group is in thereduced form.

In another embodiment, the invention provides a reagent for preparing athrombus imaging agent that is capable of being radiolabeled for imagingthrombi within a mammalian body, comprising a specific binding compoundthat specifically binds to the platelet GPIIb/IIIa receptor, and that iscovalently linked to a radiolabel-complexing moiety of formula: ##STR1##[for purposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolinic acid (Pic)-based moieties];##STR2## [for purposes of this invention, radiolabel-binding moietieshaving this structure will be referred to as picolylanine (Pica)-basedmoieties]; wherein X is H or a protecting group; (amino acid) is anyprimary α- or β-amino acid not containing a thiol group; theradiolabel-complexing moiety is covalently lined to the peptide and thecomplex of the radiolabel-complexing moiety and the radiolabel iselectrically neutral. In a preferred embodiment, the amino acid isglycine and X is an acetamidomethyl protecting group. In additionalpreferred embodiments, the peptide is covalently linked to theradiolabel-complexing moiety via an amino acid, most preferably glycine.

Yet another embodiment of the invention provides a reagent for preparinga thrombus imaging agent that is capable of being radiolabeled forimaging thrombi within a mammalian body, comprising a specific bindingcompound that specifically binds to the platelet GPIIb/IIIa receptor,and that is covalently linked to a radiolabel-complexing moiety that isa bisamino bisthiol radiolabel-complexing moiety. The bisamino bisthiolmoiety in this embodiment of the invention has a formula selected fromthe group consisting of: ##STR3## wherein each R⁵ can be independentlyH, CH₃ or C₂ H₅ ; each (pgp)^(s) can be independently a thiol protectinggroup or H; m, n and p are independently 2 or 3; A is linear or cycliclower alkyl, aryl, heterocyclyl, combinations or substituted derivativesthereof; and X is peptide; and ##STR4## wherein each R⁵ is independentlyH, lower alkyl having 1 to 6 carbon atoms, phenyl, or phenyl substitutedwith lower alkyl or lower alkoxy; m, n and p are independently 1 or 2; Ais linear or cyclic lower alkyl, aryl, heterocyclyl, combinations orsubstituted derivatives thereof; V is H or CO-(amino acid or peptide);R⁶ is H, (amino acid) or peptide; provided that when V is H, R⁶ is aminoacid or peptide and when R⁶ is H, V is amino acid or peptide, wherein(amino acid) is any primary α- or β-amino acid not containing a thiolgroup. [For purposes of this invention, radiolabel-binding moietieshaving these structures will be referred to as "BAT" moieties]. In apreferred embodiment, the peptide is covalently linked to theradiolabel-complexing moiety via an amino acid, most preferably glycine.

In preferred embodiments of the aforementioned aspects of thisinvention, the specific binding compound is a peptide comprised ofbetween 4 and 100 amino acids. The most preferred embodiment of theradiolabel is technetium-99m.

The reagents of the invention may be formed wherein the specific bindingcompounds or the radiolabel-complexing moieties are covalently linked toa polyvalent linking moiety. Polyvalent linking moieties of theinvention are comprised of at least 2 identical linker functional groupscapable of covalently bonding to specific binding compounds orradiolabel-complexing moieties. Preferred linker functional groups areprimary or secondary amines, hydroxyl groups, carboxylic acid groups orthiol-reactive groups. In preferred embodiments, the polyvalent linkingmoieties are comprised of bis-succinimidylmethylether (BSME),4-(2,2-dimethylacetyl)benzoic acid (DMAB), tris(succinimidylethyl)amine(TSEA), tris(2-chloroacetamidoethyl)amine,1,2-bis-[2-(chloroacetamido)ethoxy]ethane, andN-[2-(N',N'-bis(2-succinimidoethyl) aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazonanamide (BAT-BS).

The invention also comprises scintigraphic imaging agents that arecomplexes of the reagents of the invention with Tc-99m, ¹¹¹ In or ⁶⁸ Ga,most preferably Tc-99m and methods for radiolabeling the reagents.Tc-99m radiolabeled complexes provided by the invention are formed byreacting the reagents of the invention with Tc-99m in the presence of areducing agent. Preferred reducing agents include but are not limited todithionite ion, stannous ion and ferrous ion. Complexes of the inventionare also formed by labeling the reagents of the invention with Tc-99m byligand exchange of a prereduced Tc-99m complex as provided herein.

The invention also provides kits for preparing scintigraphic imagingagents that are the reagents of the invention radiolabeled with Tc-99m.Kits for labeling the reagents provided by the invention with Tc-99m arecomprised of a sealed vial containing a predetermined quantity of areagent of the invention and a sufficient amount of reducing agent tolabel the reagent with Tc-99m.

This invention provides methods for preparing peptide reagents of theinvention by chemical synthesis in vitro. In a preferred embodiment,peptides are synthesized by solid phase peptide synthesis.

This invention provides methods for using scintigraphic imaging agentsthat are Tc-99m labeled reagents for imaging thrombi within a mammalianbody by obtaining in vivo gamma scintigraphic images. These methodscomprise administering an effective diagnostic amount of Tc-99m labeledreagents of the invention and detecting the gamma radiation emitted bythe Tc-99m label localized at the thrombus site within the mammalianbody.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides reagents, including peptide reagents, forpreparing radiolabeled thrombus imaging agents for imaging a thrombuswithin a mammalian body. The reagents provided by the invention comprisea radiolabel binding moiety covalently linked to a specific bindingcompound that binds a platelet receptor that is the platelet GPIIb/IIIareceptor and is capable of inhibiting human platelet aggregation inplatelet-rich plasma by 50% when present at a concentration of no morethan 0.3 μM. For purposes of the invention, the term thrombus imagingreagent will refer to embodiments of the invention comprising a specificbinding compound covalently linked to a radiolabel-complexing moiety andradiolabeled, preferably with Tc-99m, ¹¹¹ In or ⁶⁸ Ga, most preferablywith Tc-99m.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Another advantage of thepresent invention is that none of the preferred radionuclides are toxic,in contrast to other radionuclides known in the art (for example, ¹²⁵I).

In the Tc-99m complexing moieties and compounds covalently linked tosuch moieties that contain a thiol covalently linked to a thiolprotecting groups [(pgp)^(s) ] provided by the invention, thethiol-protecting groups may be the same or different and may be but arenot limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

--9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Each specific-binding peptide-containing embodiment of the invention iscomprised of a sequence of amino acids. The term amino acid as used inthis invention is intended to include all L- and D, primary α- orβ-amino acids, naturally occurring and otherwise. Specific-bindingpeptides provided by the invention include but are not limited topeptides having the following sequences (the amino acids in thefollowing peptides are L-amino acids except where otherwise indicated):##STR5## and O-(4-piperdinyl)butyl tyrosine.

Specific-binding peptides of the present invention can be chemicallysynthesized in vitro. Peptides of the present invention can generallyadvantageously be prepared on an peptide synthesizer. The peptides ofthis invention can be synthesized wherein the radiolabel-binding moietyis covalently linked to the peptide during chemical synthesis in vitro,using techniques well known to those with skill in the art. Suchpeptides covalently-linked to the radiolabel-binding moiety duringsynthesis are advantageous because specific sites of covalent linkagecan be determined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodimentscomprising picolinic acid [(Pic-); e.g., Pic-GlyCys(protecting group)-],the radiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example,αN(Fmoc)-Lys-εN[Pic-Gly-Cys(protecting group)], which may beincorporated at any position in the peptide chain. This sequence isparticularly advantageous as it affords an easy mode of incorporationinto the target binding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety[-Cys(protecting group)-Gly-Pica] can be prepared during peptidesynthesis by including the sequence [-Cys(protecting group)-Gly-] at thecarboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys- and-Cys-Gly-Pica chelators are provided in the Examples hereinbelow. Thisinvention provides for the incorporation of these chelators intovirtually any peptide capable of specifically binding to a thrombus invivo, resulting in a radiolabeled peptide having Tc-99m held as neutralcomplex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m. This invention provides for the incorporation of thesechelators into virtually any peptide capable of specifically binding toa thrombus in vivo, resulting in a radiolabeled peptide having Tc-99mheld as neutral complex. An example of a small synthetic peptidecontaining a BAT chelator as radiolabel-binding moiety is provided inthe Examples hereinbelow.

In forming a complex of radioactive technetium with the reagents of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the reagent in the presence of a reducingagent. Preferred reducing agents are dithionite, stannous and ferrousions; the most preferred reducing agent is stannous chloride. Means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apreformed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts. In a preferredembodiment of the invention, a kit for preparing technetium-labeledpeptides is provided. An appropriate amount of the reagent is introducedinto a vial containing a reducing agent, such as stannous chloride, inan amount sufficient to label the reagent with Tc-99m. An appropriateamount of a transfer ligand as described (such as tartrate, citrate,gluconate or mannitol, for example) can also be included. The kit mayalso contain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The components of the kitmay be in liquid, frozen or dry form. In a preferred embodiment, kitcomponents are provided in lyophilized form.

Radiolabeled thrombus imaging reagents according to the presentinvention may be prepared by the addition of an appropriate amount ofTc-99m or Tc-99m complex into the vials and reaction under conditionsdescribed in Example 4 hereinbelow.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

The thrombus imaging reagents provided by the present invention can beused for visualizing thrombi in a mammalian body when Th-99m labeled. Inaccordance with this invention, the Tc-99m labeled reagents areadministered in a single unit injectable dose. The Tc-99m labeledreagents provided by the invention may be administered intravenously inany conventional medium for intravenous injection such as an aqueoussaline medium, or in blood plasma medium. Generally, the unit dose to beadministered has a radioactivity of about 0.01 mCi to about 100 mCi,preferably 1 mCi to 20 mCi. The solution to be injected at unit dosageis from about 0.01 mL to about 10 mL. After intravenous administration,imaging of the thrombus in vivo can take place in a matter of a fewminutes. However, imaging can take place, if desired, in hours or evenlonger, after the radiolabeled peptide is injected into a patient. Inmost instances, a sufficient amount of the administered dose willaccumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

It will also be recognized by those having skill in the relevant artsthat thrombi are commonly found at sites of atherosclerotic plaque; thatintegrin receptors that may bind to the scintigraphic imaging agents ofthe invention may be found in certain tumors; and that such integrinreceptors are involved in cell adhesion processes that accompany orinitiate leukocyte localization at sites of infection. Therefore it willbe recognized that the scintigraphic imaging agents of this inventionhave additional utility as imaging agents for imaging sites in which theGPIIb/IIIa receptor is expressed, including atherosclerotic plaques,tumors and sites of infection.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Synthesis of BAT-BM(N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropl)-6,9-diazanonanamide)

BAT-BM was prepared as follows. BAT acid (N⁹ -(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanoicacid)(10.03g, 10.89 mmol) and 75 mL of dry methylene chloride (CH₂ Cl₂) were addedto a 250 mL round-bottomed flask equipped with magnetic stir bar andargon balloon. To this solution was added diisopropyl carhodiide (3.40mL, 21.7 mmol, 199 mole %), followed by N-hydroxy-succinimide (3.12 g,27.1 mmol, 249 mole %). This solution was observed to become cloudywithin 1 h, and was further incubated with stirring for a total of 4 hat room temperature. A solution of tris(2-aminoethyl)amine (30 mL, 200mmol, 1840 mole %) in 30 mL methylene chloride was then added andstirring continued overnight. The reaction mixture was then concentratedunder reduced pressure, and the residue partitioned between ethylacetate(150 mL) and 0.5 M potassium carbonate (K₂ CO₃ ; 150 mL). The organiclayer was separated, washed with brine and concentrated to give thecrude product N-[2-(N',N'-bis(2-aminoethyl)amino ethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl-6,9-diazanonanamide as afoam/oil.

This crude product was added to a 1000 mL round-bottomed flask, equippedwith magnetic stir bar, containing 300 mL THF, and then 30 mL saturatedsodium bicarbonate (NaHCO₃), 100 mL water and N-methoxycarbonylmaleimide (6.13 g, 39.5 mmol, 363 mole %) were added. This heterogeneousmixture was stirred overnight at room temperature. THF was removed fromthe mixture by rotary evaporation, and the aqueous residue was twiceextracted with ethylacetate (2×75 mL). The combined organic layers ofthese extractions were washed with brine, dried over sodium sulfate,filtered through a medium frit and concentrated to about 12 g of crudeproduct. Purification by liquid chromatography (250 g silicondioxide/eluted with a gradient of chloroform→2% methanol in chloroform)afforded 5.3 g of pure product(N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide)(equivalent to 40% yield), along with approximately 5 g of crude productthat can be repurified to yield pure product. Chemical analysis of thepurified product confirmed its identity as BAT-BM as follows:

¹ H NMR (200 mHz, CDCl₃): δ0.91 (12H,s), 1.38 (9H,s), 1.2-1.6 (4H,m),2.06 (2H,s), 2.18 (2H,t,J=7), 2.31 (4H,m), 2.55 (2H,t,J=5), 2.61(4H,t,J=6), 2.99 (2H,s), 3.0-3.3 (4H,m), 3.46 (4H,t,J=6), 6.49(--NH,t,J=4), 6.64 (4H,s), 7.1-7.3 (18H,m), 7.6 (12H,t,J=17).

EXAMPLE 2 Synthesis of TMEA [tris(2-maleimidoethyl)amine]

tris(2-aminoethyl)amine (1.49 mL, 10 mmol) dissolved in 50 mL saturatedaqueous sodium bicarbonate and cooled in an ice bath, was treated withN-carbomethoxymaleimide (4.808 g, 31 mmol). The mixture was stirred for30 min on ice and then for another 30 min at room temperature. Themixture was then partitioned between dichloromethane and water, driedover magnesium sulfate, filtered and evaporated to give 3.442 g ofproduct. Reverse phase thin-layer chromatography (RP-TLC) yieldedessentially 1 spot (R_(f) =0.63 in 1:1 acetonitrile:0.5 M sodiumchloride). 3.94 mmol (1.817 g) of this product was dissolved in 20 mLtetrahydrofuran and 20 mL saturated sodium bicarbonate and mixed for 2h. The reaction mixture was then partitioned between ethyl acetate andwater. The organic phase was washed with saturated sodium chloride,dried over magnesium sulfate, and filtered. The ethyl acetate solutionwas diluted with hexanes and cooled. Solid TMEA was collected byfiltration and dried to a yield of 832 mg. Chemical analysis of theproduct confirmed its identity as TMEA as follows:

¹ H NMR (CDCl₃): 2.65 (tr. 2H), 3.45 (tr.2H). 6.64 (s. 2H). ¹³ C NMR(CDCl₃), 35.5, 51.5, 133.9, 170.4.

EXAMPLE 3 Synthesis of tris[2-(2-chloroacetamido)ethyl]amine

tris[2-(2-chloroacetamido)ethyl]amine was prepared as follows. To asolution of tris(2-aminoethyl)amine (1.49 mL, 10 mmol) indichloromethane (50 mL) was added powdered K₂ CO₃ (6.9 g, 50 mmol)followed by chloroacetyl chloride (2.6 mL, 33 mmol) dropwise. Vigorousboiling resulted. The mixture was cooled and 50 mL water added. Theorganic layer was separated, dried over MgSO₄ and evaporated to give thetitle compound as a crystalline solid (2.5 g, 6.6 mmol, equal to 66%yield). Thin layer chromatography, performed using C18 reverse phasechromatography plates and a 50:50 mixture of acetonitrile and 0.5 M NaClin water, showed a single spot with R_(f) equal to 0.62. The meltingpoint of the crystals of the title compound so produced was determinedto be between 116-119° C.

EXAMPLE 4 Synthesis of 1,2-bis2-(2-chloroacetamido)ethoxylethane

1,2-bis[2-(2-chloroacetamido)ethoxy]ethane was prepared as follows. To amixture of 1,2-bis(2-aminoethoxy)ethane (1.5 g, 10 mmol) indichloromethane (100 mL) and 1 M Na₂ CO₃ (50 mL) cooled in an ice bathwas added chloroacetyl chloride (2.6mL, 33 mmol) dropwise. Afterstirring for 10 minutes, the organic layer was separated and evaporatedto give the title compound (2.6 g, eual to 92% yield). HLPC analysisrevealed a single peak using ultraviolet (220 nm) absorbtionspectrometry.

EXAMPLE 5 Synthesis ofNε-(Nα,Nε-bis-chloroacetyllysyl)lysyl-glycyl-(S-trityl)cysteinamide

Nε-(Nα,Nε-bis-chloroacetyllysyl)lysyl-glycyl-(S-trityl)cysteinamide wasprepared as follows. The protected peptide was synthesized using SPPSmethodology as described in Example 7 using Rink amide resin,(Fmoc)Cys(Trt), (Fmoc)Gly, (Boc)Lys(Fmoc), (Fmoc)Lys(Fmoc) andchloroacetic acid. The peptide was cleaved from the resin, deprotectedand re-S-tritylated to give the title compound which was then purifiedby reverse phase HPLC. Synthesis was confirmed using FABMS; MH⁺ wasfound to be 828 and 830 for a peptide having a theoretical molecularweight of 829 (average).

EXAMPLE 6 Synthesis of Nα-(Fmoc)-4-(N-pentamethylchromansulfonyl)amidinophenylalanine [Fmoc-Amp(Pmc)]

Title compound Fmoc-Amp(Pmc) was prepared as follows.D,L-acetyl-4-cyanophenylalanine was prepared from α-bromotoluonitrileand diethyl acetamidomalonate using conventional techniques (see Marvel,1955, Org. Syn. Cell. 3: 705-708) and resolved by selective hydrolysisof the L-epimer using hog renal acylase (see Greenstein et al., 1961, inChemistry of the Amino Acids, Kreiger & Malabar, eds., pp. 2172-2174).

L-4-cyanophenylalanine was converted to the N-α-Boc, t-butyl ester usingconventional techniques. The N-α-Boc-4-cyanophenylalanine, t-butyl esterwas converted into the 4-amidino derivative using the method of Voigt etal. (1988, Pharmazie 43: 412-414). The resultingN-α-Boc4-amidinophenylalanine, t-butyl ester was converted into theN-amidinopentamethylchromansulfonyl (Pmc) derivative essentially asdescribed for arginine (see Ramage et al., 1987, Tetrahedron Lett. 28:2287-2290). The Boc and t-butyl groups were then removed usingBF₃.OEt/acetic acid. The resulting4-pentamethylchromasulfonyl-amidinophenylalanine was converted to thetitle compound using N-(fluorenylmethoxycarboxy)succinimide (FmocOSu).

The title compound was analyzed by HPLC and found to have a retentiontime of 10.4 minutes using a waters Nova-Pak C18 reverse phase radialcompression column and eluted at 3 mL/min with a gradient of 100% (0.1%TFA/water) to 100% (0.1% TFA/10% water/acetonitrile) over 10 minutes.The title compound was analyzed by ¹ H NMR spectrometry and found tohave the following ¹ H NMR spectrum (sample dissolved in CDCl₃): δ1.25(s, 6H), 1.72 (t, 2H), 2.1 (s, 3H), 2.55 (m, 8H), 3.1 (m, 2H), 4.05-4.7(m, 4H), 5.5 (d, 1H) and 7.0-8.0 (m, 15H).

EXAMPLE 7 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotmazole or2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid or 50/50 trifluoroaceticacid/dichloromethane, optionally containing water, thioanisole,ethanedithiol, and triethylsilane, prepared in ratios of 100:5:5:2.5:2for 1.5-3 h at room temperature.

Where appropriate, N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. For preparingbranched-chain peptide reagents involving peptide chain synthesis fromboth the α- and ε-amines of lysine, Nα(Fmoc)Nε(Fmoc)-lysine was usedduring SPPS. Where appropriate, 2-chloroacetyl and 2-bromoacetyl groupswere introduced either by using the appropriate 2-halo-acetic acid asthe last residue to be coupled during SPPS or by treating the N-terminusfree amino peptide bound to the resin with either the 2-halo-aceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP of the2-halo-acetic anhydride/diisopropylethylamine in NMP. Where appropriate,HPLC-purified 2-haloacetylated peptides were cyclized by stirring in a0.1-1.0 mg/mL solution at pH8 optionally containing phosphate,bicarbonate or 0.5-1.0 mM EDTA for 0.5-48 hours, followed byacidification with acetic acid, lyophilization and HPLC purification.Where appropriate, Cys-Cys disulfide bond cyclizations were performed bytreating the precursor cysteine-free thiol peptides at 0.1 mg/mL in pH 7buffer with aliquots of 0.006 M K₃ Fe(CN)₆ until a stable yellow colorpersisted. The excess oxidant was reduced with excess cysteine, themixture was lyophilized and then purified by HPLC.

Where appropriate, peptide thiol-chloroacetyl derived sulfides wereprepared by reacting single thiol-containing peptides at a concentrationof 2 to 50 mg/mL in water and acetonitrile or THF or DMF at pH 10 withthe appropriate number (e.g., 0.5 molar equivalents for preparing dimersand 0.33 molar equivalents for preparing trimers) of the chloroacetylpolyvalent linker moiety for 0.5 to 24 hours. The solution was thenneutralized with acetic acid, evaporated to dryness, and, if necessary,deprotected using 10 mL TFA and scavengers such as 0.2 mL triethylsilanefor 30 to 90 minutes. The solution was concentrated and the product wasprecipitated with ether. Products were purified by preparative HPLC.

Where appropriate, BSME adducts were prepared by reacting singlethiol-containing peptides (5 to 50 mg/mL in 50 mM sodium phosphatebuffer, pH 7 to 8) with 0.5 molar equivalents of BMME(bis-maleimidomethylether) pre-dissolved in acetonitrile at roomtemperature for approximately 1-18 hours. The solution was concentratedand the product was purified by HPLC.

Where appropriate, TSEA adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 10 to 100 mg/mL peptidein DMF, or 5 to 50 mg/mL peptide in 50 mM sodium phosphate (pH8)/acetonitrile or THF) with 0.33 molar equivalents of TMEA(tris(2-maleimidoethyl)amine; Example 2) pre-dissolved in acetonitrileor DMF, with or without 1 molar equivalent of triethanolamine, at roomtemperature for approximately 1-18 h. Such reaction mixtures containingadducts were concentrated and the adducts were then purified using HPLC.

Where appropriate, BAT-BS adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 2 to 50 mg/mL peptide in50 mM sodium phosphate (pH 8)/acetonitrile or THF) with 0.5 molarequivalents of BAT-BM (N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide;Example 1) pre-dissolved in acetonitrile or THF, at room temperature forapproximately 1-18h. The solution was then evaporated to dryness and[BAT-BS]-peptide conjugates deprotected by treatment with 10 mL TFA and0.2 mL triethylsilane for 1 h. The solution was concentrated, theproduct adducts precipitated with ether, and then purified by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS) or electrospray massspectroscopy (ESMS).

EXAMPLE 8 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer, 0.1 M bicarbonate buffer or10% hydroxypropylcyclodextrin (HPCD), each buffer at pH of 5-10. Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E. I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using thefollowing conditions: a Waters DeltaPure RP-18, 5μ, 150 mm×3.9 mmanalytical column was loaded with each radiolabeled peptide and thepeptides eluted at a solvent flow rate equal to 1 mL/min. Gradientelution was performed beginning with 10% solvent A (0.1% CF3COOH/H₂ O)to 40% solvent B₉₀ (0.1% CF₃ COOH/90% CH₃ CN/H₂ O) over the course of 20min.

The Tc-99m labeled peptide purity was determined by HPLC using theconditions described in the Footnotes in Table I. Radioactive componentswere detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 1 using the method described herein.

                                      TABLE I                                     __________________________________________________________________________                                          FABMS                                                                              Radiochemical                                                                          HPLC                      Peptides                              MH.sup.+                                                                           Yield (%)*                                                                             R.sub.T                   __________________________________________________________________________                                                        (min)**                   CH.sub.2 CO.Y.sub.D RGDCC.sub.Acm GC.sub.Acm amide.sup.b  (SEQ ID NO.:        8)                                    1057 97.sup.2 10.0, 10.4,                                                                   10.6.sup.2                CH.sub.2 CO.Y.sub.D RGDCGGC.sub.Acm GC.sub.Acm amide (SEQ ID NO.:                                                   1171 99.sup.2 13.5.sup.2                CH.sub.2 CO.Y.sub.D.Apc.GDCGGGC.sub.Acm GC.sub.Acm amide (SEQ ID NO.:                                               1233 100.sup.4                                                                              17.1, 18.1.sup.2          GRGDVRGDFKC.sub.Acm GC.sub.Acm amide (SEQ ID NO.: 11)                                                               1510 97.sup.2 16.2, 16.8.sup.2          GRGDVRGDFC.sub.Acm GC.sub.Acm amide (SEQ ID NO.: 12)                                                                1382 94.sup.2 16.4.sup.2                CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGF.sub.D PRPG.NH.sub.2      (SEQ ID NO.: 13)                     1845 90.sup.4 16.6, 16.9.sup.2          (CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2                                                 3020.sup.a                                                                         98.sup.4 9.3.sup.2                 (CH.sub.2 CO.Y.sub.D.Apc.GDCggc.sub.Acm GC.sub.Acm GGC.amide).sub.3                                                 4596A                                                                              99.sup.4 9.2, 11.6.sup.5           (CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2           -[BAT-BS]                             3409.sup.a                                                                         98.sup.3 10.3.sup.5                C.sub.Acm GC.sub.Acm RRRRRRRRRGDV (SEQ ID NO.: 14)                                                                  2100 100.sup.2                                                                              2.4.sup.3***              (CH.sub.2 CO.Y.sub.D Apc.GDCKGC.sub.Acm GC.sub.Acm GGC.amide).sub.2                                                 3163.sup.a                                                                         98.sup.4 9.6.sup.5                 (CH.sub.2 COY.sub.D.Apc.GDCGGC.sub.Acm GC.sub.Acm GGCamide).sub.2             -K(Nε-K)GCamide               3298.sup.a                                                                         96.sup.3 12.0.sup.4                (CH.sub.2 COY.sub.D.Amp.GDCGGC.sub.Acm GC.sub.Acm GGCamide).sub.2             -(CH.sub.2 CO).sub.2 K(Nε-K)GCamide                                                                         3357.sup.a                                                                         99.sup.8 4.6.sup.6                 (CH.sub.2 COY.sub.D.Amp.GDCKGCGamide).sub.2 -(CH.sub.2 CO).sub.2 K(N.epsil    on.-K)GCamide                         2573.sup.a                                                                         98.sup.8 4.8.sup.6                 __________________________________________________________________________     *Superscripts refer to the following labeling conditions:                     .sup.1 The peptide was dissolved in 50 mM potassium phosphate buffer (pH      7.4) and labeled at room temperature.                                         .sup.2 The peptide was dissolved in 50 mM potassium phosphate buffer (pH      7.4) and labeled at 100° C.                                            .sup.3 The peptide was dissolved in water and labeled at room temperature     .sup.4 The peptide was dissolved in water and labeled at 100° C.       .sup.5 The peptide was dissolved in 50 mM potassium phosphate buffer (pH      6.0) and labeled at 100° C.                                            .sup.6 The peptide was dissolved in 50 mM potassium phosphate buffer (pH      5.0) and labeled at room temperature.                                         .sup.7 The peptide was dissolved in 50:50 mixture of ethanol/water and        labeled at 100° C.                                                     .sup.8 The peptide was dissolved in 0.9% sodium chloride solution and         labeled at room temperature.                                                  **HPLC methods (indicated by superscript after R.sub.T):                      general:                                                                      solvent A = 0.1% CF.sub.3 COOH/H.sub.2 O                                      solvent B.sub.70  = 0.1% CF.sub.3 COOH/70% CH.sub.3 CN/H.sub.2 O              solvent B.sub.90  = 0.1% CF.sub.3 COOH/90% CH.sub.3 CN/H.sub.2 O              sovent flow rate = 1 mL/min                                                   Vydak column = Vydak 218TP54 RP18, 5 νm, 220 mm x 4.6 mm analytical        column with guard column                                                      Brownlee column = Brownlee Spheri5 RP18, 5 μm, 220 mm x 4.6 mm column      Waters column = Waters DeltaPak C18, 5μm, 150 mm x 3.9 mm column           Waters column 2 = Waters NovaPak C18, 5μm, 100 mm x 8 mm radial            compression column                                                            Method 1: Brownlee column 100% A to 100% B.sub.70  in 10 min                  Method 2: Vydak column 100% A to 100% B.sub.90  in 10 min                     Method 3: Vydak column 100% A to 100% B.sub.70  in 10 min                     Method 4: Waters column 100% A to 100% B.sub.90  in 20 min                    Method 5: Waters column 100% A to 100% B.sub.90  in 10 min                    Method 6: Waters 2 column 100% A to 100% B.sub.90  in 10 min                  ***Confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis      Singleletter abbreviations for amino acids can be found in G. Zubay,          Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33;           underlining indicates the formation of a thiol linkage between the linked     amino acids of derivative groups; peptides are linked to BSH, ETAC, BSME,     TSEA, [BATBS] OR (CH.sub.2 CO)containing linkers via the free thiol moiet     of the unprotected cysteine residue (C) in each peptide;                      Ac = acetyl;                                                                  Bz = benzoyl;                                                                 Pic = picolinoyl (pyridine2-carbonyl);                                        Acm = acetamidomethyl;                                                        Mob = 4methoxybenzyl;                                                         Apc = L[S(3-aminopropyl)cysteine;                                             Hly = homolysine;                                                             F.sub.D = Dphenylalanine;                                                     Y.sub.D = Dtyrosine;                                                          ma = 2mercaptoacetic acid;                                                    mmp = 2mercapto-2-methylpropionic acid;                                       BAT = N.sup.6,N.sup.9 bis(2mercapto-2-methylpropyl)-6,9-diazanonanoic         acid;                                                                         ETAC = 4(O-CH.sub.2 COGly-Gly-Cys.amide)acetophenone;                         BATBS = N[2N',Nbis(2succinimidoethyl)aminoethylN.sup.6,N.sup.9                bis(2mercapto-2-methylpropyl)-6,9-diazanonanamide;                            BSME = bissuccinimidomethylether;                                             TSEA = tris(2-succinimidoethyl)amine;                                         NES = Nethylsuccinimide;                                                      BSH = 1,6bissuccinimidohexane;                                                Amp = 4amidinophenylalanine                                                   .sup.a confirmed by electrospray mass spectrometry [ESMS                 

EXAMPLE 9 Platelet Aggregation Inhibition Assays

Platelet aggregation studies were performed essentially as described byZucker (1989, Methods in Enzymol. 169: 117-133). Briefly, plateletaggregation was assayed with or without putative platelet aggregationinhibitory compounds using fresh human platelet-rich plasma, comprising300,000 platelets per microliter. Platelet aggregation was induced bythe addition of a solution of adenosine diphosphate to a finalconcentration of 10 to 15 micromolar, and the extent of plateletaggregation monitored using a Bio/Data aggregometer (Bio/Data Corp.,Horsham, Pa.). The concentrations of platelet aggregation inhibitorycompounds used were varied from 0.1 to 500 μg/mL. The concentration ofinhibitor that reduced the extent of platelet aggregation by 50%(defined as the IC₅₀) was determined from plots of inhibitorconcentration versus extent of platelet aggregation. An inhibition curvefor peptide RGDS was determined for each batch of platelets tested.

The results of these experiments are shown in Table II. In Table II, thecompounds tested are as follows (RGDS is given as a positive control):

P47=AcSYGRGDVRGDFKC_(Acm) GC_(ACm) (SEQ ID NO.:15)

P97=GRGDVRGDFKC_(Acm) GC_(Acm) amide (SEQ ID NO.:11)

P32=C_(Acm) GC_(Acm) RRRRRRRRRGDV (SEQ ID NO.:14)

P143=CH₂ CO-Y_(D) RGDCGGC_(Acm) GC_(Acm) amide (SEQ ID NO.:9)

P245=CH₂ CO-Y_(D),Apc,AGDCGGC_(Acm) GC_(Acm) GGF_(D) PRPGamide (SEQ IDNO.:13)

P63=AcSYGRGDVRGDFKCTCCA (SEQ ID NO.:16)

P98=GRDGVRGDFC_(Acm) GC_(Acm) amide (SEQ ID NO.:12)

P81=CH₂ CO-Y_(D) RGDCC_(Acm) GC_(Acm) amide (SEQ ID NO.:8)

P154=CH₂ CO-Y_(D) ApcGDGGGC_(Acm) GC_(Acm) amide (SEQ ID NO.:10)##STR6##

These results demonstrate that the IC₅₀ decreases for cyclic peptides ascompared with linear ones, and is even less for polyvalent peptideagents as compared with monovalent peptide agents. These resultsillustrate the efficacy of the multimeric polyvalent antithromboticagents of the invention at reducing platelet aggregation.

                  TABLE II                                                        ______________________________________                                        Peptides     IC.sub.50 (μM)**                                                                     Clot/Blood*                                            ______________________________________                                        P357         0.079     6.3 ± 3.4.sup.6                                     P667         0.081     5.9, 5.0.sup.2                                         P280         0.090     4.4 ± 1.8.sup.5                                     P682         0.130     4.0.sup.1                                              P317         0.036     3.8 ± 2.2.sup.3                                     P381         0.035     2.5                                                    P154         0.3       2.0 ± 0.5.sup.3                                     P245         0.5       1.5                                                    P143         1.3       1.4                                                    P97          8         1.0                                                    P98          15        1.7                                                    P63          19        1.7                                                    P47          23        1.0                                                    P81          25        1.8 ± 0.6.sup.3                                     P32          26        1.2 ± 0.2.sup.4                                     ______________________________________                                         .sup.1 n = 1;                                                                 .sup.2 n = 2;                                                                 .sup.2 n = 3;                                                                 .sup.4 n = 4;                                                                 .sup.5 n = 6;                                                                 .sup.6 n = 9                                                                  *ratio of (% injected dose/g in a femoral vein thrombus)/(% injected          dose/g in blood) at approximately 4 h postinjection of each Tc99 m labele     reagent in a canine model of DVT                                              **concentration of reagent that inhibits by 50% the aggregation of human      platelets in plateletrich plasma induced to aggregate by the addition of      adenosine diphosphate (ADP)                                              

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:   16                                          - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Tyr Xaa Gly Asp Cys Gly Gly Gly                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Tyr Xaa Gly Asp Cys Lys Gly                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Tyr Xaa Gly Asp Cys Gly Gly                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Tyr Xaa Gly Asp Cys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Tyr Xaa Gly Asp Cys Lys                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Amp) OTHER INFORMATION:                                              #"Residue Xaa = 4-amidinophenylalanine."                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Tyr Xaa Gly Asp Cys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Amp) OTHER INFORMATION:                                              #"Residue Xaa = 4-amidinophenylalanine."                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Tyr Xaa Gly Asp Cys Lys                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6..8                                                  #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Tyr Arg Gly Asp Cys Cys Gly Cys                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8..10                                                 #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Tyr Arg Gly Asp Cys Gly Gly Cys Gly Cys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9..11                                                 #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Tyr Xaa Gly Asp Cys Gly Gly Gly Cys Gly Cy - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11..13                                                #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Gly Arg Gly Asp Val Arg Gly Asp Phe Lys Cy - #s Gly Cys                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10..12                                                #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Gly Arg Gly Asp Val Arg Gly Asp Phe Cys Gl - #y Cys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= D-TyrOTHER INFORMATION:                                              #"The tyrosine residue is in the D-stereo-                                    #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Apc) OTHER INFORMATION:                                              #"Residue Xaa = L(S-3 aminopropyl)                                                           cysteine."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..5                                                  #/label= CyclicTHER INFORMATION:                                              #"The sidechain sulfur of the Cys                                                            residue i - #s covalently linked to the amino                  CH2CO- group." terminus                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8..10                                                 #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= D-PheOTHER INFORMATION:                                              #"The phenylalanine residue is in the D-stereo-                               #configuration"chemical                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Tyr Xaa Gly Asp Cys Gly Gly Cys Gly Cys Gl - #y Gly Phe Pro Arg Pro         Gly                                                                           #                15                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1..3                                                  #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= AmideOTHER INFORMATION:                                              #"The carboxyl terminus is modified to an                                                    amide"                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Cys Gly Cys Arg Arg Arg Arg Arg Arg Arg Ar - #g Arg Gly Asp Val             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= AcetylTHER INFORMATION:                                              #"The amino terminus is modified with an                                                     acetyl gr - #oup."                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13..15                                                #/label= Tc-99m-chelatorRMATION:                                              #"The sidechain sulfur atoms of both Cys                                      #are each protected with an                                                                  acetamidomet - #hyl group"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Ser Tyr Gly Arg Gly Asp Val Arg Gly Asp Ph - #e Lys Cys Gly Cys             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= AcetylTHER INFORMATION:                                              #"The amino terminus is modified with an                                                     acetyl gr - #oup."                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Ser Tyr Gly Arg Gly Asp Val Arg Gly Asp Ph - #e Lys Cys Thr Cys Cys         Ala                                                                           #                15                                                           __________________________________________________________________________

What is claimed is:
 1. A reagent for preparing a thrombus imaging agentcomprising, in combination, a technetium-99m complexing moietycovalently linked to a platelet glycoprotein IIb/IIIa receptor bindingmoiety having a molecular weight of less than 10,000 daltons, whereinthe reagent inhibits human platelet aggregation in platelet-rich plasmaby 50% (IC₅₀) when the reagent is present at a concentration of no morethan 0.3 μM.
 2. The reagent of claim 1, wherein the receptor bindingmoiety is a peptide comprising from 4 to 100 amino acids.
 3. The reagentof claim 1, wherein the technetium-99m complexing moiety has a formulaselected from the group consisting of:

    Cp(aa)Cp

wherein Cp is a protected cysteine and (aa) is any primary α- or β-aminoacid not containing a thiol group; ##STR7## wherein X=H or a protectinggroup; (amino acid)=any primary α- or β-amino acid not containing athiol group; ##STR8## wherein X=H or a protecting group; (aminoacid)=any primary α- or β-amino acid not containing a thiol group;##STR9## wherein each R⁵ is independently H, CH₃ or C₂ H₅ ; each(pgp)^(s) is independently a thiol protecting group or H; m, n and p areindependently 2 or 3; A=linear lower alkyl, cyclic lower alkyl, aryl,heterocyclyl, or a combination thereof; and ##STR10## wherein each R⁵ isindependently H, lower alkyl having 1 to 6 carbon atoms, phenyl, orphenyl substituted with lower alkyl or lower alkoxy; m, n and p areindependently 1 or 2; A=linear lower alkyl, cyclic lower alkyl, aryl,heterocyclyl, or a combination thereof; V=H or --CO-peptide; R⁶ =H orpeptide;and wherein when V=H, R⁶ =peptide and when R⁶ =H,V=--CO-peptide.
 4. The reagent of claim 2, wherein the peptide and thetechnetium-99m complexing moiety are covalently linked through one ormore amino acids.
 5. The reagent of claim 3, wherein the technetium-99mcomplexing moiety has the formula

    Cp(aa)Cp

and Cp comprises a protecting group having a formula

    --CH.sub.2 --NH--CO--R

wherein R is a lower alkyl having 1 to 6 carbon atoms, 2-pyridyl,3-pyridyl, 4-pyridyl, phenyl, or phenyl substituted with lower alkyl,hydroxy, lower alkoxy, carboxy, or lower alkoxycarbonyl.
 6. The reagentof claim 3, wherein the technetium-99m complexing moiety has theformula: ##STR11##
 7. A scintigraphic imaging agent comprising thereagent of claim 1, wherein the technetium-99m complexing moiety iscomplexed with technetium-99m.
 8. The reagent of claim 2, wherein thepeptide is selected from the group consisting of:
 9. A complex formed byreacting the reagent of claim 1 with technetium-99m in the presence of areducing agent.
 10. The complex of claim 9, wherein the reducing agentis selected from the group consisting of a dithionite ion, a stannousion, and a ferrous ion.
 11. A kit for preparing a radiopharmaceuticalpreparation, said kit comprising a sealed vial containing apredetermined quantity of the reagent of claim 1 and a sufficient amountof a reducing agent to label the reagent with technetium-99m.
 12. Aprocess of preparing the reagent of claim 2 by chemical in vitrosynthesis.
 13. The process of claim 12, wherein the synthesis is solidphase peptide synthesis.
 14. A method of labeling the reagent of claim1, comprising the step of reacting the reagent with technetium-99m inthe presence of a reducing agent.
 15. The method of claim 14, whereinthe reducing agent is selected from the group consisting of a dithioniteion, a stannous ion, and a ferrous ion.
 16. The reagent of claim 2,wherein the peptide comprises a cyclic domain having a formula: whereinA is a lipophilic D-α-amino acid, an N-alkyl-L-α-amino acid orL-proline;X is an L-α-amino acid having a positively charged sidechain;and R is each independently H, lower alkyl or lower alkoxyalkyl.
 17. Thereagent of claim 16, wherein A is D-tyrosine or D-phenylalanine and X isL-{S-(3-aminopropyl)cysteine} or L-4-amidinophenylalanine.
 18. Thereagent of claim 1, wherein the technetium-99m complexing moietycomprises a single thiol-containing moiety of formula:

    A-CZ(B)-{C(R.sup.1 R.sup.2)}.sub.n -X                      II.

wherein A is H, HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acidor peptide)-OOC or R⁴ ; B is H, SH, --NHR³, --N(³)-(amino acid orpeptide), or R⁴ ; X is H, SH, --NHR³, --N(R³)-(amino acid or peptide) orR⁴ ; Z is H or R⁴ ; R¹, R², R³ and R⁴ are independently H, lowerstraight chain alkyl, lower branched chain alkyl, or cyclic alkyl; n is0, 1, or 2;and where B is --NHR³ or --N(R³)-(amino acid or peptide), Xis SH, and n is 1 or 2; where X is --NHR³ or --N(R³)-(amino acid orpeptide), B is SH, and n is 1 or 2; where B is H or R⁴, A is HOOC, H₂NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC, X is SH,and n is 0 or 1; where A is H or R⁴, then where B is SH, X is --NHR³ or--N(R³)-(amino acid or peptide) and where X is SH, B is --NHR³ or--N(l³)-(amino acid or peptide); where X is H or R⁴, A is HOOC, H₂ NOC,(amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and B is SH;where Z is methyl, X is methyl, A is HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC, B is SH and n is 0;andwherein (amino acid) is any primary α- or β-amino acid not containing athiol group.
 19. The reagent of claim 18, wherein the technetium-99mcomplexing moiety is selected from the group consisting of:-(aminoacid)¹ -(amino acid)² -{A-CZ(B)-(C(R¹ R²))_(n) -X}, -{ A-CZ(B)-(C(R¹R²))_(n) -X}-(amino acid)¹ -(amino acid)², -(a primary α,ω- orβ,ω-diamino acid)-(amino acid)¹ -{A-CZ(B)-(C(R¹ R²))_(n) -X} and-{A-CZ(B)-(C(R¹ R²))_(n) -X}-(amino acid)¹ -(a primary α,ω- orβ,ω-diamino acid).
 20. A reagent for preparing a thrombus imaging agentcomprising:a) a polyvalent linker; b) at least two platelet glycoproteinIIb/IIIa receptor binding compounds, each compound being covalentlylinked to the linker; and c) at least two radiolabel complexingmoieties, each moiety being covalently linked to the linker;said reagenthaving a molecular weight of less than about 20,000 daltons; wherein thereagent inhibits human platelet aggregation in platelet-rich plasma by50% (IC₅₀) when the reagent is present at a concentration of no morethan 0.3 μM.
 21. The reagent of claim 20, wherein the linker isbis-succinimidylmethylether, 4-(2,2-dimethylacetyl)benzoic acid,N-[2-(N',N'bis(2-succinimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonamide,tris(succinimidylethyl)amine, tris(2-chloroacetamidoethyl) amine, or1,2-bis(2-chloroacetamidoethoxy)ethane.
 22. A method of imaging athrombus within a mammalian body comprising the steps of:a)administering an effective diagnostic amount of the agent of claim 7 tosaid body; and b) detecting technetium-99m accumulated at the thrombus.23. A composition comprising a reagent having a formula: ##STR12##
 24. Acomposition comprising a reagent having a formula:
 25. A compositioncomprising a reagent having a formula: